CCDC109B CRISPR Activation Plasmid (h): sc-412415-ACT

MCUB (CCDC109B) encodes a regulatory subunit of the mitochondrial calcium uniporter complex that modulates Ca2+ influx across the inner mitochondrial membrane and helps tune mitochondrial bioenergetics and Ca2+-dependent signaling. By acting as a negative regulator of MCU channel activity, CCDC109B influences oxidative phosphorylation, reactive oxygen species balance, and coupling between cytosolic Ca2+ transients and mitochondrial metabolism. Altered uniporter regulation has been linked to dysregulated cell stress responses and remodeling of mitochondrial dynamics, processes relevant to proliferative and neurodegenerative phenotypes. In human cells, MCUB/CCDC109B is therefore studied in the context of mitochondrial Ca2+ homeostasis, metabolic reprogramming, and Ca2+-driven transcriptional programs.

CCDC109B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MCUB expression without altering the underlying DNA sequence.

CCDC109B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MCUB locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the MCUB transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CCDC109B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MCUB locus and enabling the study of CCDC109B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CCDC109B pathway restoration in tumor cells with silenced or reduced MCUB expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.